Liquid Biopsy in Organ Damage: small extracellular vesicle chip-based assessment of polytrauma

Background Despite major advances in medicine, blood-borne biomarkers are urgently needed to support decision-making, including polytrauma. Here, we assessed serum-derived extracellular vesicles (EVs) as potential markers of decision-making in polytrauma. Objective Our Liquid Biopsy in Organ Damage (LiBOD) study aimed to differentiate polytrauma with organ injury from polytrauma without organ injury. We analysed of blood-borne small EVs at the individual level using a combination of immunocapture and high-resolution imaging. Methods To this end, we isolated, purified, and characterized small EVs according to the latest Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines from human blood collected within 24 h post-trauma and validated our results using a porcine polytrauma model. Results We found that small EVs derived from monocytes CD14+ and CD14+CD61+ were significantly elevated in polytrauma with organ damage. To be precise, our findings revealed that CD9+CD14+ and CD14+CD61+ small EVs exhibited superior performance compared to CD9+CD61+ small EVs in accurately indicating polytrauma with organ damage, reaching a sensitivity and a specificity of 0.81% and 0.97%, respectively. The results in humans were confirmed in an independent porcine model of polytrauma. Conclusion These findings suggest that these specific types of small EVs may serve as valuable, non-invasive, and objective biomarkers for assessing and monitoring the severity of polytrauma and associated organ damage.

2) were added, and cells were incubated for an additional 15 min in the dark on ice.Designated antibodies were added according to the manufacturer's recommendations and titrated against their matching isotype controls (Suppl.Table 2).The cells were then incubated in the dark on ice for 15 min.Subsequently, 3 μL of 7-AAD (BD Pharmingen, New Jersey, USA, 559925) was added and cells were incubated in the dark at 4°C for another 5 min.The cells were analyzed on a BD FACSCanto II flow cytometer (BD Biosciences, New Jersey, USA).All experiments were performed in technical and biological triplicates.

Isolation and purification of THP-1 derived small EVs by size-exclusion chromatography (SEC) and ultrafiltration (UF) from cell culture supernatant
When cells have reached at least 50% confluence, cell culture supernatants were harvested and centrifuged at 500 × g for 10 min to remove cells, cell debris, and larger apoptotic bodies.This was followed by centrifugation at 10,000 × g for 10 min to remove the large EVs.The Large EV depleted supernatant was then transferred to a 100 kDa MWCO filter (Amicon Ultra, Merck, Darmstadt, Germany) and concentrated to a volume of 2 mL at room temperature (RT).The sample was loaded onto a qEV2/35 SEC column (Izon Science, Christchurch, New Zealand), and the buffer flow volume of 14.1 mL was discarded, after which the following five fractions (2 mL each) were collected.The collected fractions were concentrated to 1 mL using a 3 kDa MWCO filter (Amicon Ultra, Merck, Darmstadt, Germany) and stored at -80°C until further use.

Isolation of THP-1 derived large EVs by centrifugation from cell culture supernatant
Large EVs were isolated according to previously pre-established protocols (2)(3)(4).The supernatants of LPS-induced macrophages were centrifuged at 300 × g for 5 min at 4 °C to remove cells, cell debris, and larger apoptotic bodies.The supernatant was further purified by centrifugation at 2,000 x g for 30 min, followed by centrifugation at 20,000 × g for 1 h at 4 °C.Resulting large EV-enriched pellets were resuspended in filtered PBS, pH 7.4 (0.22 µm) (Merck Millipore, Boston, USA) and stored at -80℃ until usage.

Isolation of serum from human blood
Blood sampling was carried out in accordance with the recommendations of the European Liquid Biopsy Society (ELBS) and followed the ISO 20186-1:2019 'Molecular in vitro diagnostic examinations -Specifications for pre-examination processes for venous whole blood.' ISO 20186 contains specific guidelines on the processing of venous blood and small EVs.Serum samples were obtained from polytrauma patients with an ISS ≥ 15. 10 mL of whole blood was collected from a venous or arterial access post-traumatic.Blood samples were collected within 24 hrs and 7 days post-trauma.Full-blood was centrifuged at 3000 × g for 3 min at RT, and the supernatants were collected.Sera were stored permanently at -80 °C.For the shipment of human specimens, the following regulations were strictly obeyed as UN 2814, UN 3291, or UN 3373 (UN Recommendations on the Transport of Dangerous Goods) without interrupting the freezing chain.

Transmission electron microscopy of EVs
The isolated EV samples were diluted 100-fold with ultrapure water.7.5 µL of the sample solution was mixed with 1 µL of aqueous contrast solution containing 1% methyl cellulose (w/v; Sigma Aldrich, Darmstadt, Germany) and 2% uranyl acetate (w/v; Polysciences, Warrington, USA).After incubation for 10 min, a 0.5 µL droplet was placed on a 200-mesh copper grid covered with a carbon-coated Formvar film (Plano, Wetzlar, Germany) and dried at room temperature to allow EVs to adhere to the film surface.Images were taken using a JEM 1400Plus electron microscope (JEOL, Tokyo, Japan) at 120 kV, equipped with a 4096 × 4096 pixel CMOS camera (TemCam-F416; TVIPS, Gauting, Germany).The EMMENU image acquisition software (version 4.09.83) was used to obtain 16-bit images.

Western blot analysis
Lysed samples were treated with the reducing agent dithiothreitol (DTT) to resolve the proteins according to their molecular weight.ProteinSimple capillary immunoassay (Wes) (ProteinSimple, Bio-Techne, Minneapolis, MN, USA) was performed with 2 μg of protein per capillary according to the manufacturer's instructions (5).The following primary antibodies were used for WES, as summarized in Suppl.Table 3.

Multiple trauma model -porcine study a) Instrumentation and Anaesthesia
The porcine model has been described in details elsewhere (6)(7)(8).Therefore, only a short summary of the model is elucidated within this publication.
Before start of experiment, pigs completed a 12 hrs fasting period, with water ad libitum.On the day of operation, premedication (azaperone four mg/kg body weight) was administered by an intramuscular injection.During operation and observation time anaesthesia was maintained through continuous 1% propofol (Fresenius, Germany), midazolam (Panpharma, Germany) and fentanyl (Panpharma, Germany) administration.Ventilation with lung protective parameters during observation period (8 -12 mL/kg/body weight, 50% oxygen) was carried out by usage of a Draeger Evita (Lübeck, Germany) ventilation device.
In order to maintain physiological homeostasis and nutrition in pigs continous aminoven (370 kcal/L 50 -70 mL/kg body weight and day) and crystalloid infusions (Sterofundin ISO®; 0.5 -2.0 mL/kg/hrs) were administered.These fluids and medication were administered through two central venous catheters (Arrow Medical, UK) placed into right femoral vein and external jugular vein.Mean arterial blood pressure (MAP) was measured via an arterial line (Vygon, Germany) placed into left femoral artery and monitored via a Philips patient monitor (Philips Health Systems, Hamburg, Germany).Finally, a suprapubic catheter was placed into the bladder.Pigs were warmed with air blowers to a body temperature of 38.5 -39.5°C.

b) Trauma and Haemorrhage
After instrumentation, stable baseline conditions were reached after 120 min.Subsequently, oxygen supply was set to 21%, warming and previous continued fluid administration was put to 0.1 mL/hrs to keep the transfusion lines open but not to provide further fluid supply and trauma was induced and maintained for 90 min (trauma phase).
During trauma phase, polytrauma pigs (PT) received a blunt chest trauma with a bolt gun (Blitz-Kerner, turbocut JOBB GmbH, Germany) fired at a double layer panel of 1 cm lead on top and 1 cm steel.The same device was used to induce femur fractures on both sides using a T-shaped steel panel.Furthermore, a midline mini-laparotomy was performed to explore right hepatic lobe.Standardized liver laceration in combination with pressure controlled haemorrhagic shock (40±5 mm HG) were induced for 90 min via blood drawing through venous catheter.
After trauma phase resuscitation of pigs including reperfusion of blood and fluids, FiO2 levels of 50 %, warming and administration of antibiotics (Ceftriaxon® 2 g i.v./every 24 hrs) were performed regarding established trauma guidelines (ATLS®, AWMF-S3 guideline on Treatment of Patients with Severe and Multiple Injuries®).The resuscitation phase was followed by surgical phase (OP phase; 60 min).

c) Group allocation
Altogether, 8 pigs were stabilized with external fixation (Radiolucent Fixator, Orthofix), and 8 pigs with non-reaming intra medullary nailing (T2 System, Stryker) following the DCO and ETC guideline procedures.6 pigs were allocated to the Sham group; therefore, no intervention other than general instrumentation and anaesthesia was performed.

Plasma isolation from full-blood -porcine study
The EDTA K blood plasma samples (Sarstedt, Nümbrecht, Germany, S-Monovette) were centrifuged at 2000 g for 15 min at 4 °C immediately after blood draw within the operation theatre to separate blood cells from plasma.Subsequently, plasma was filtered through a 0.22 μm syringe filter (Sartorius, Göttingen, Germany Cat# 16532) to remove cell debris.Plasma was stored at -80˚C for further use, and no additional melting or freezing cycles were added prior to EV isolation.

Supplement Figures and Tables
Suppl. Figure 1: Extended Polytrauma (ISS ≥ 15) Patient's demographics.Overview of given medications/drugs as indicted including pre-medication taken by patients on a daily base pre-trauma as Aspirin and overview of blood parameters as indicated.Correlation calculation between depicted parameters and their use is depicted in Suppl.Table 2.
Suppl. Figure 2: Representative ExoView® Tetraspanin spots -cell culture derived small EVs.Depicted are exemplary original composite color dot spot images for tetraspanins CD9 + (blue) and CD63 + (red) and CD81 + (green) small EVs purified from culture as captured by anti-CD9, anti-CD63 and anti-CD81 capture spots on ExoView® Tetraspanin Chips with lab internal number 008 and corresponding isotype MIgG composite image as control for unspecific small EV capture.Depicted are all spots as measured by ExoView® Reader R100 which are selected and analyzed by ExoView® Analysis software automatically.
Suppl. Figure 4: Representative ExoView® Tetraspanin spots -human serum derived small EVs.Depicted are exemplary original composite colour dot spot images for CD14 + (green) and CD61 + (red) and CD14 + CD61 + (yellow) small EVs purified from human serum as captured by anti-CD9, anti-CD63 and anti-CD81 capture spots on ExoView® Tetraspanin Chips with lab internal number 018 and corresponding isotype MIgG composite image as control for unspecific small EV capture.Depicted are all spots as measured by ExoView® Reader R100 which are selected and analysed by ExoView® Analysis software.MIgG CHIP018.010 was disabled by analysis software and designated as SPOT DISABLED in red.

Supplement Tables
Suppl.Suppl.Table 2 Overview of antibodies used for FACS analysis.Designated antibodies were added according to manufacturer's recommendations and antibodies were titrated against their matching isotype controls prior use.

Figure 5 :
small EV release on investigated time axis (day 7 post trauma) and correlations with days of invasive ventilation and duration of ICU.(A) and (B) depicts Spearman correlation analysis for median CD9 + CD14 + small EVs and CD9 + CD61 + small EV values with associated patient's days of invasive ventilation (n = 48).(C) Spearman correlation analysis for median CD9 + CD14 + small EVs values and (D) CD9 + CD61 + small EV values associated with patients' ICU duration (n = 47).P -and rsp values as indicated.Suppl.Figure 6: Representative ExoFlex® Tetraspanin spots -porcine plasma derived small EVs.Exemplary original color dot spot images are shown for CD14 + (in green) particles isolated by SEC and UF from porcine plasma as captured by anti-CD9 and stained for CD14.MIgG as unspecific small EV binding control.Depicted are all spots on ExoFlex® chip with lab internal number 53 as measured by ExoView® Reader R100 which are analyzed by ExoView® Analysis software.

Table 4
Overview of antibodies for ExoView® Reader R100

Table 5
Summarized data values of individual small EV populations in polytrauma including associated SEM, fold change and statistics